Antibodies are powerful tools for the detection, monitoring and quantitation of an unknown antigen in a sample wherein immunoassays have acquired widespread use. Enzyme immunosorbent immunoassays, typically referred to as an ELISA, are the most common form of immunoassays wherein a primary or secondary antibody is labeled with an enzyme or enzyme binding molecule wherein a luminescent signal is obtained after binding of the antigen. This method is heterogeneous and requires multiple steps and reagents. In the past decade fluorescent labeled primary and secondary antibodies have been employed for the detection of antigen allowing for more sensitivity and detection in samples such as tissue sections and intracellular targets. A particular type of immunoassay includes competitive immunoassays wherein the presence and amount of a particular antigen in an unknown sample is determined by its ability to compete with labeled reference antigen for binding to an antibody immobilized on a solid surface, e.g., microwell plate. To quantitate the bound antigen a standard curve of known concentrations of the antigen must be assayed with the unknown samples. Competitive immunoassays are powerful tools for their ability to detect small quantities of antigen in a complex sample, however, due to inherent limitations they have not acquired widespread use. Herein we report a novel homogenous competitive assay that allows for rapid detection of a target ligand in a sample.
The present invention takes advantage of fluorescent-labeled ligand analogs and fluorophore or quencher labeled labeling proteins wherein a target ligand displaces the ligand analog resulting in a change in the detectable signal. The change in detectable signal can be a result of FRET, inherent quenching by the ligand-binding antibody or the use of a fluorogenic ligand analog. Competitive immunoassays using chromophores as quenchers and acceptors for FRET have been disclosed wherein the ligand analog is conjugated to a chromophore and the primary or secondary antibody is conjugated to another chromophore (U.S. Pat. Nos. 3,998,943; 4,160,016; 4,174,384; 4,261,968; 3,996,345). This method has limitations in that either the primary antibody or secondary antibody are required to be conjugated to a fluorophore. When the primary antibody is conjugated to a chromophore the labeling method often result in labeling that interferes with binding or a lower detectable signal than expected. In addition, the labeling of primary antibody is an additional step for the end user. Alternatively, labeling of secondary antibody results in a more universal immunoassay, however the secondary antibody needs to be added sequentially to the sample or risk the primary+secondary antibody complex precipitating out of solution.
We have developed a homogenous competitive immunoassay that does not require labeling of primary antibody or the use of a labeled secondary antibody. For this invention a labeling protein is employed that is a monovalent antibody fragment or non-antibody protein that can be pre-complexed with the primary antibody. In addition, the present invention takes advantage of the inherent quenching of the ligand analog by the ligand antibody that is accomplished by appropriate matching of the ligand analog and the ligand-binding antibody.